DESCRIPTION (Applicant's Description): Integral membrane proteins pose a unique challenge to traditional methods of structure determination by virtue of the fact that they are present in a membrane. As a result, many researchers are reluctant to address the problems associated with membrane protein structure determination because of the extensive time commitment and the level of risk involved. More than half of the almost 500 targets for which the pharmaceutical industry has developed molecules that alleviate disease fall into a class of integral membrane proteins known as G-protein coupled receptors. Many of these receptors play key roles in cardiovascular disease, diabetes, hypertension, AIDS, and a variety of sensory and mental disorders. It is because of the significant difficulty and lack of established methodology for the expression, purification, and crystallization of G-protein coupled receptors that the development and application of additional, alternative approaches for their high-resolution structure determination is proposed. Specifically, the goals of this research are to provide solutions to the aforementioned concerns using the G-protein coupled receptor's rhodopsin and CCR5 as models. Recent observations from this and other laboratories on the remarkable propensity of G-protein coupled receptor fragments to fold and assemble in an autonomous manner suggests that the cytoplasmic, membrane-embedded, and extracellular regions of these receptors can be regarded as independent domains. Further, biochemical studies on various soluble polypeptide subdomains of the cytoplasmic surface of rhodopsin show that they effectively mimic the signaling functions of the activated receptor. Initial efforts will focus on the construction of molecular models for these receptors by combining high resolution structural information obtained through NMR or crystallographic analysis of sections of the surface and transmembrane domains along with distance constraints derived from crosslinking studies aimed at determining the nearest neighbor organization of the transmembrane spans. A three-part, multidisciplinary approach to solve the three-dimensional structures of the intact receptors by X-ray crystallography will also be investigated. This will involve the large-scale expression and purification of these receptors or their mutants, the use of antibody fragments or soluble protein targets as exogenous "hydrophilic domains" to promote receptor crystallization, and the analysis of bicontinuous lipidic cubic phases for their ability to provide a suitable hydrophobic environment for the nucleation and growth of well-ordered crystals. Collectively, it is anticipated that these approaches will provide a framework for determining high-resolution structures of entire families of membrane proteins and will ultimately allow a more rational approach to the design of drugs for these essential targets.